Hanna Edlund

Origin of the U87MG glioma cell line: Good news and bad news

The DNA profile of the publicly available and widely used glioma cell line U87MG is quite different from that of the original cell line. U87MG: Not what it used to be A cell line commonly used for research on gliomas is found to be different from the original tumor from which it was derived. The authors, whose laboratory developed the cell line almost 50 years ago, compared the genetics of this line (obtained from ATCC) with those of the original tumor. They report that the DNA profile of the current cell line differs from that of the original cells but that it is likely to be a human glioblastoma cell line with unknown origins. This misidentification of a widely studied cell line reinforces the need for researchers to carefully validate the cell lines used in their research. Human tumor–derived cell lines are indispensable tools for basic and translational oncology. They have an infinite life span and are easy to handle and scalable, and results can be obtained with high reproducibility. However, a tumor-derived cell line may not be authentic to the tumor of origin. Two major questions emerge: Have the identity of the donor and the actual tumor origin of the cell line been accurately determined? To what extent does the cell line reflect the phenotype of the tumor type of origin? The importance of these questions is greatest in translational research. We have examined these questions using genetic profiling and transcriptome analysis in human glioma cell lines. We find that the DNA profile of the widely used glioma cell line U87MG is different from that of the original cells and that it is likely to be a bona fide human glioblastoma cell line of unknown origin.

Southern African ancient genomes estimate modern human divergence to 350,000 to 260,000 years ago

Ancient DNA pushes human emergence back Anatomically modern humans evolved in Africa, but pinpointing when has been difficult. Schlebusch et al. sequenced three ancient African genomes from the Stone Age, about 2000 years old, and four from the Iron Age, 300 to 500 years old. One of the oldest samples, sequenced to 13× coverage, appears most closely to resemble individuals from the present-day San population. However, this individual seems to have lacked genetic contributions from other modern African populations, including pastoralists and farmers, which were observed in modern San individuals. Thus, the earliest divergence between human populations may have occurred 350,000 to 260,000 years ago. Science, this issue p. 652 Ancient African genomes push back the timing of the split between human populations. Southern Africa is consistently placed as a potential region for the evolution of Homo sapiens. We present genome sequences, up to 13x coverage, from seven ancient individuals from KwaZulu-Natal, South Africa. The remains of three Stone Age hunter-gatherers (about 2000 years old) were genetically similar to current-day southern San groups, and those of four Iron Age farmers (300 to 500 years old) were genetically similar to present-day Bantu-language speakers. We estimate that all modern-day Khoe-San groups have been influenced by 9 to 30% genetic admixture from East Africans/Eurasians. Using traditional and new approaches, we estimate the first modern human population divergence time to between 350,000 and 260,000 years ago. This estimate increases the deepest divergence among modern humans, coinciding with anatomical developments of archaic humans into modern humans, as represented in the local fossil record.

Forensic analysis of autosomal STR markers using Pyrosequencing.

Short tandem repeats (STRs) are highly variable, and therefore routinely used in forensic investigations for a DNA-based individual identification. The routine assay is commonly performed by size separation using capillary electrophoresis, but alternative technologies can also be used. In this study, a Pyrosequencing assay was developed for analysis of STR markers useful in forensic DNA analysis. The assay was evaluated for 10 different STR loci (CSF1PO, TH01, TPOX, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539 and Penta E) and a total of 114 Swedish individuals were genotyped. This genotyping strategy reveal the actual sequence and variant alleles were seen at several loci, providing additional information compared to fragment size analysis. At the D13S317 locus a T/A SNP located in the last repeat unit was observed in 92% of the genotypes. Moreover, an upstream flanking SNP at locus D7S820, a SNP within the repeats at D3S1358 and D8S1179 and a deletion in the flanking region at locus D5S818 were observed. The Pyrosequencing method was first developed for SNP typing and sequencing of shorter DNA fragments but the method also provides an alternative method for STR analysis of less complex repeats. This assay is suitable for investigation of new markers, a rapid compilation of population data and for confirmation of variant and new alleles.

Long-term genetic stability and a high-altitude East Asian origin for the peoples of the high valleys of the Himalayan arc

Significance Since prehistory, the Himalayan mountain range has presented a formidable barrier to population migration, whereas at the same time its transverse valleys have long served as conduits for trade and exchange. Yet, despite the economic and cultural importance of Himalayan trade routes, little is known about the region’s peopling and early population history. In this study, we conduct to our knowledge the first ancient DNA investigation of the Himalayan arc and generate genome data for eight individuals ranging in time from the earliest known human settlements to the establishment of the Tibetan Empire. We demonstrate that the region was colonized by East Asians of likely high-altitude origin, followed by millennia of genetic continuity despite marked changes in material culture and mortuary behavior. The high-altitude transverse valleys [>3,000 m above sea level (masl)] of the Himalayan arc from Arunachal Pradesh to Ladahk were among the last habitable places permanently colonized by prehistoric humans due to the challenges of resource scarcity, cold stress, and hypoxia. The modern populations of these valleys, who share cultural and linguistic affinities with peoples found today on the Tibetan plateau, are commonly assumed to be the descendants of the earliest inhabitants of the Himalayan arc. However, this assumption has been challenged by archaeological and osteological evidence suggesting that these valleys may have been originally populated from areas other than the Tibetan plateau, including those at low elevation. To investigate the peopling and early population history of this dynamic high-altitude contact zone, we sequenced the genomes (0.04×–7.25×, mean 2.16×) and mitochondrial genomes (20.8×–1,311.0×, mean 482.1×) of eight individuals dating to three periods with distinct material culture in the Annapurna Conservation Area (ACA) of Nepal, spanning 3,150–1,250 y before present (yBP). We demonstrate that the region is characterized by long-term stability of the population genetic make-up despite marked changes in material culture. The ancient genomes, uniparental haplotypes, and high-altitude adaptive alleles suggest a high-altitude East Asian origin for prehistoric Himalayan populations.

Analysis of the Putative Remains of a European Patron Saint–St. Birgitta

Saint Birgitta (Saint Bridget of Sweden) lived between 1303 and 1373 and was designated one of Europes six patron saints by the Pope in 1999. According to legend, the skulls of St. Birgitta and her daughter Katarina are maintained in a relic shrine in Vadstena abbey, mid Sweden. The origin of the two skulls was assessed first by analysis of mitochondrial DNA (mtDNA) to confirm a maternal relationship. The results of this analysis displayed several differences between the two individuals, thus supporting an interpretation of the two skulls not being individuals that are maternally related. Because the efficiency of PCR amplification and quantity of DNA suggested a different amount of degradation and possibly a very different age for each of the skulls, an orthogonal procedure, radiocarbon dating, was performed. The radiocarbon dating results suggest an age difference of at least 200 years and neither of the dating results coincides with the period St. Birgitta or her daughter Katarina lived. The relic, thought to originate from St. Birgitta, has an age corresponding to the 13th century (1215–1270 cal AD, 2σ confidence), which is older than expected. Thus, the two different analyses are consistent in questioning the authenticity of either of the human skulls maintained in the Vadstena relic shrine being that of St. Birgitta. Of course there are limitations when interpreting the data of any ancient biological materials and these must be considered for a final decision on the authenticity of the remains.

An Analysis of the Alleged Skeletal Remains of Carin Göring

In 1991, treasure hunters found skeletal remains in an area close to the destroyed country residence of former Nazi leader Hermann Göring in northeastern Berlin. The remains, which were believed to belong to Carin Göring, who was buried at the site, were examined to determine whether it was possible to make a positive identification. The anthropological analysis showed that the remains come from an adult woman. The DNA analysis of several bone elements showed female sex, and a reference sample from Carins son revealed mtDNA sequences identical to the remains. The profile has one nucleotide difference from the Cambridge reference sequence (rCRS), the common variant 263G. A database search resulted in a frequency of this mtDNA sequence of about 10% out of more than 7,000 European haplotypes. The mtDNA sequence found in the ulna, the cranium and the reference sample is, thus, very common among Europeans. Therefore, nuclear DNA analysis was attempted. The remains as well as a sample from Carins son were successfully analysed for the three nuclear markers TH01, D7S820 and D8S1179. The nuclear DNA analysis of the two samples revealed one shared allele for each of the three markers, supporting a mother and son relationship. This genetic information together with anthropological and historical files provides an additional piece of circumstantial evidence in our efforts to identify the remains of Carin Göring.

Y chromosomal STR analysis using Pyrosequencing technology

Analysis of Y chromosome STR markers has proven to be useful in forensic cases where the samples contain a mixture of DNA from several individuals. STR markers are commonly genotyped based on length separation of PCR products. In this study we evaluated if Pyrosequencing can be used as an alternative method for determining Y-STR variants. In total 70 unrelated Swedish males were typed for the Y chromosomal markers (DYS19, DYS389 I-II, DYS390, DYS391, DYS392, DYS393 and DYS438) using Pyrosequencing. Using the 8 markers, 57 unique haplotypes were observed with a discrimination capacity of 0.81. At four loci, the Pyrosequencing analysis revealed sequence variants. The sequence variants were found in the DYS389 II, DYS390, DYS391, and DYS393 loci in frequencies between 1.43% and 14.3%. Pyrosequencing has here been shown to be a useful tool for typing Y chromosomal STRs and the method can provide a complement to conventional forensic Y STR analyses. Moreover, the Pyrosequencing method can be used to rapidly evaluate novel markers.

Ancient genomes from southern Africa pushes modern human divergence beyond 260,000 years ago

Southern Africa is consistently placed as one of the potential regions for the evolution of Homo sapiens. To examine the region’s human prehistory prior to the arrival of migrants from East and West Africa or Eurasia in the last 1,700 years, we generated and analyzed genome sequence data from seven ancient individuals from KwaZulu-Natal, South Africa. Three Stone Age hunter-gatherers date to ~2,000 years ago, and we show that they were related to current-day southern San groups such as the Karretjie People. Four Iron Age farmers (300–500 years old) have genetic signatures similar to present day Bantu-speakers. The genome sequence (13x coverage) of a juvenile boy from Ballito Bay, who lived ~2,000 years ago, demonstrates that southern African Stone Age hunter-gatherers were not impacted by recent admixture; however, we estimate that all modern-day Khoekhoe and San groups have been influenced by 9–22% genetic admixture from East African/Eurasian pastoralist groups arriving >1,000 years ago, including the Ju|‘hoansi San, previously thought to have very low levels of admixture. Using traditional and new approaches, we estimate the population divergence time between the Ballito Bay boy and other groups to beyond 260,000 years ago. These estimates dramatically increases the deepest divergence amongst modern humans, coincide with the onset of the Middle Stone Age in sub-Saharan Africa, and coincide with anatomical developments of archaic humans into modern humans as represented in the local fossil record. Cumulatively, cross-disciplinary records increasingly point to southern Africa as a potential (not necessarily exclusive) ‘hot spot’ for the evolution of our species.

Genomics of Mesolithic Scandinavia reveal colonization routes and high-latitude adaptation

Scandinavia was one of the last geographic areas in Europe to become habitable for humans after the last glaciation. However, the origin(s) of the first colonizers and their migration routes remain unclear. We sequenced the genomes, up to 57x coverage, of seven hunter-gatherers excavated across Scandinavia and dated to 9,500-6,000 years before present. Surprisingly, among the Scandinavian Mesolithic individuals, the genetic data display an east-west genetic gradient that opposes the pattern seen in other parts of Mesolithic Europe. This result suggests that Scandinavia was initially colonized following two different routes: one from the south, the other from the northeast. The latter followed the ice-free Norwegian north Atlantic coast, along which novel and advanced pressure-blade stone-tool techniques may have spread. These two groups met and mixed in Scandinavia, creating a genetically diverse population, which shows patterns of genetic adaptation to high latitude environments. These adaptations include high frequencies of low pigmentation variants and a gene-region associated with physical performance, which shows strong continuity into modern-day northern Europeans.

Forensic Analysis of Mitochondrial and Autosomal Markers Using Pyrosequencing

Forensic casework analyses often face challenges, such as limited genetic material with or without fragmentation and damage. To compensate for low amounts and degradation, shorter amplicons are often applied in the analysis. Also, a change of markers might be necessary using mitochondrial instead of autosomal markers. In addition, forensic research often involves analysis of large number of samples for marker evaluation and population-database compilation. Therefore, a flexible, robust but also rapid method for the detection of variation is highly useful. Pyrosequencing(®) is a rapid, reliable, easy-to-use method for sequence analysis. The method is well suited for rapid forensic analysis of a few targets or analysis of a single target in many samples. It allows sequencing of very short amplicons, which facilitates analysis of degraded DNA. Here we present the use of Pyrosequencing, a robust method for sensitive forensic analysis of mitochondrial DNA, autosomal STRs, and Y-chromosome STRs and SNPs.